Cardio-cerebrovascular diseases are number one killer threatening human health and life, and thrombosis is an important cause of many cardio-cerebrovascular diseases. Therefore, anticoagulants are important medicines used to prevent thrombosis. At present, heparin is an anticoagulant extensively used in clinical treatment. But one of its important shortcomings is to initiate thrombocytopenia. The newly developed Low-Molecular-Weight (LMW) heparin might decrease above risks, but can not thoroughly overcome its shortcomings.
People have focused more and more on above-mentioned shortcomings of prior anticoagulants. As an ideal anticoagulant, it should have the clear and definite anticoagulant effect, and should not cause the hemorrhagic side-effect in the case of systemic administration, so as to increase the clinical safety.
For this purpose, the key point of guiding principles for the present invention is that the anticoagulant function should be conditionally and specifically activated. That is, this kind of material normally has no anticoagulant activity, and only when the coagulation system is activated, and thrombus is possible to form or the thrombus formed, then this kind of anticoagulant locally display their anticoagulant activities. The local anticoagulant activity around the developing or developed thrombus forms a microenviroment to prevent thrombosis or the continued growing of the thrombus, even to dissolve the formed minute thrombus so as to attain the prophylactic/therapeutic purposes. Therefore, this invention would overcome the risk of systemic hemorrhage which often happens with administration of anticoagulant agents, such as heparin, hirudin etc.
For example, hirudin is a single chain polypeptide consisting of 65-66 amino acid residues, whose amino terminus can bind with the catalytic active site of thrombin, and possesses anticoagulant activity, and the binding of its C-terminus with the recognition site of thrombin substrate shows a very strong specific affinity to thrombin. This study designed a measure to block the amino-terminus of hirudin so as to diminish hirudin's anticoagulant activity temporarily. When the in vivo coagulation system is activated and thrombus formed, the special biochemical changes incited by thrombosis make the amino terminal-blocked hirudin recovered to the original form of hirudin displaying the specific anticoagulation effect at the location of potential or occurred thrombosis, thereby the systemic hemorrhagic side effect could be decreased. Therefore, this is a new type of safe and effective anticoagulant agent.
Under guidance of the above-mentioned inventive idea, hirudin was modified in this study. Hirudin first was linked by its amino-terminal with an oligopeptide recognizable and cleavable by thrombin, which is named as TH, or with an oligopeptide recognizable and cleavable by blood coagulation factor Xa, which is named as FH, so that once the coagulation system was activated, the anticoagulant activity of hirudin derivatives could be released and play the anticoagulant and antithrombotic roles, while the hemorrhagic side-effect would be also decreased. The results indicated that the modified hirudin was functional, but the effectiveness was not still high enough. Then, we developed our idea and considered that the hirudin might be modified by linking with an oligopeptide which can be recognized and cleaved by several blood coagulation factors or other factors. We hoped that the effectiveness could be enhanced by the above-mentioned modification to the extent of applicability to clinical practice. At present, however, on the basis of the related references, we can not infer if these oligopeptides can be recognized and cleaved by the corresponding blood coagulation factors in high efficiency. Two related proteins were prepared in our laboratory as follows: (1) hirudin was linked at its amino-terminal with the oligopeptide being recognizable and cleavable by both thrombin and coagulation factor Xa, this hirudin derivative being named as GH; (2) hirudin was linked at its amino-terminal with the oligopeptide being recognizable and cleavable by both coagulation factors XIa and Xa, this hirudin derivative being named as EH.
The research results indicated that these two derivatives of hirudin normally had no anticoagulation activity whether in vitro or in vivo, and could effectively release anticoagulation activity of hirudin locally under coacting of the above mentioned blood coagulation factors once coagulation system being activated, blood coagulation factors and play both roles of anticoagulation and antithrombosis. Certainly, There was no anticoagulation activity in the site of the body where thrombosis was absent, and thus their systemic hemorrhagic side-effect was also notably decreased. Therefore, unlike anticoagulant, such as hirudin and heparin, they are a kind of safe, effective anticoagulant and antithrombotic agents. As a result, this kind of anticoagulant agents with the characteristics of low hemorrhage is of importance to the application in prevention and/or treatment of thrombosis.